Muteins of placental growth factor type 1, preparation method and application thereof

ABSTRACT

The present invention relates to chemically stable muteins of type 1 Placental Growth Factor (PLGF-1) bearing the substitution or elimination of a cysteine residue from the wild type protein amino acid sequence, their preparation, their therapeutic and cosmetic uses, and pharmaceutical and cosmetic compositions containing said derivatives. The invention likewise relates to the production of antibodies for said derivatives and their use in the diagnosis and treatment of tumoral and non-tumoral pathologies.

FIELD OF THE INVENTION

The present invention relates to stable muteins of type 1 PlacentalGrowth Factor (PLGF-1), their preparation, their therapeutic andcosmetic use and pharmaceutical and cosmetic compositions containingsaid derivatives. The invention likewise relates to the production ofantibodies to said derivatives and their use in the diagnosis andtreatment of tumoral and non-tumoral pathologies.

STATE OF THE ART

Type 1 Placental Growth Factor (PLGF-1) is an angiogenic homodimericglycoprotein. Angiogenic activity relates to the dimeric form, as themonomeric form is inactive. The complete polynucleotide sequenceencoding the PLGF-1 protein, along with its polypeptide sequence, weredescribed by Maglione and Persico in Pat. EP-B-0 550 519(WO-A-92/06194).

The above patent describes a method for producing PLGF-1 in bacteriamodified using an inducible expression system, said method involving,after induction, bacterial lysis and direct extraction of the rawprotein from the lysate solution. The protein obtained in this way showslow levels of biological activity.

A method for extraction and purification of the raw placental factor,obtained by expression in bacteria, is described by Maglione et. al. inpatent application PCT/IT02/00065. The method involves a series ofextraction, renaturation and purification passages, which as a wholemake it possible to obtain the pure protein in an essentially dimericform, that is to say in its most active form. It is, in fact, known thatthe monomeric form of the protein is biologically inactive, and onlyacquires angiogenic functions after renaturation to the dimeric form.

However, the present inventors have observed that the protein in dimericform is partially unstable, and gives rise, in an aqueous solution,during storage or processing, to multimeric forms that show lessbiological activity and for this reason are less suitable fortherapeutic use, due to the uncertainty of doses and biologicalactivity.

The aim of the present invention is therefore to solve the problem ofpoor chemical/biological stability of PLGF-1 as observed mainly when thelatter is conserved in aqueous solutions.

SUMMARY OF THE INVENTION

The invention is based on the unexpected discovery that derivatives ofthe natural protein PLGF-1 with modifications in their polypeptidesequence, specifically involving substitution or elimination of at leastone cysteine residue, show greatly increased chemical stability, whileat the same time maintaining their original biological activityessentially unchanged. In the light of this discovery, a first object ofthe application is represented by a mutein of the monomeric form of type1 human or animal Placental Growth Factor (PLGF-1) comprisingsubstitution or elimination in the wild type protein polypeptidesequence of at least one of the nine cysteine residues (Cys) containedtherein. Said substitution or elimination does not affect thedimerisation process essential to obtain the protein in its biologicallyactive form, but prevents multimerisation of the monomeric form.

Among the various cysteine residues, it has been seen that eliminationor substitution of a residue present in the C-terminal portion, andspecifically in position 142 of the complete polypeptide sequence, thatis to say comprising both the mature PLGF-1 protein and thecorresponding signal peptide, is particularly effective. In thepreferred embodiment of the invention the residue Cys 142 is replaced bya glycine residue (Gly). This substitution produces a mutein of themonomeric form of PLGF-1 that has unchanged dimerisation capacity, butis basically incapable of generating multimerisation products. As wellas the modifications described above, the muteins according to theinvention may contain further eliminations, substitutions or additionsof one or more wild type protein amino acids, providing saidmodifications do not alter the functional characteristics of the muteinitself.

A second object of the invention is therefore represented by a mutein ofthe factor PLGF-1 in dimeric form, preferably purified in such a way asessentially to comprise the dimer alone. Said mutein may equally well bethe mature protein or the pre-protein, comprising a signal peptide inthe N-terminal portion.

A further object of the invention is the nucleotide sequence comprisingthe DNA encoding the mutein in suit. The sequence is characterised inthat a TGC or TGT codon encoding the amino acid cysteine in the naturalPLGF-1 sequence is eliminated or modified. In a preferred embodiment ofthe invention the codon corresponding to cysteine is substituted by aGGC, GGT, GGA or GGG codon, all encoding the amino acid glycine (Gly).It is preferably the thymidine base (T) in position 382 (TGC codon) ofsequence SEQ ID NO:1 that is replaced by the guanidine base (G) togenerate the GGC codon.

A further object of the invention is an expression system comprising thenucleotide sequence seen above, flanked by untranslated sequencescontrolling and regulating expression. This system can be induced inprokaryotic cells, preferably bacterial cells.

Expression is under the control of an inducible promoter and can beinduced by means of suitable compounds. Host cells modified using theexpression system seen above are also the object of the invention. Theseare prokaryotic cells, preferably bacterial cells such as E. coli. Theinvention also covers processes for production of the nucleotidesequence, in which the DNA encoding the mutein is produced by polymerasechain reaction (PCR) using as a primer oligonucleotides that have beensuitably modified with respect to the wild type protein sequence.Preferably the oligonucleotide of SEQ ID NO:3 is used as 5′-3′forwardprimer and the oligonucleotide of sequence SEQ ID NO:4 is used as5′-3′reverse primer.

A further object of the invention is a process for production andextraction of the mutein in which host cells, preferably bacterial,modified using the expression system according to the invention, arecultivated in a suitable culture medium, expression of the protein isinduced by a suitable inducer, the cells are isolated and lysated andthe mutein is extracted from the lysis mixture. During the fermentationstep and before the induction of the expression step, the cells arecultivated until a high optical density (O.D.) of the culture medium isreached. Expression of the protein is subsequently induced by additionof suitable induction agents. During the subsequent steps the cells arelysated, to release the endocellular materials into the culture medium,specifically nucleic material and inclusion bodies, the latter aresolubilised and the protein obtained in this way is renatured in dimericform. The extraction and purification process may comprise additionaloptional steps for purification of the dimeric protein. In a preferredembodiment the process comprises at least one additional purificationstep on ionic exchange or reverse phase chromatography. In a furtherembodiment the process comprises an initial anionic exchangechromatography purification followed by reverse phase chromatography.

The mutein obtained using the production, extraction and purificationprocess according to the invention contains not less than 98.5% activeprotein and not more than 1.5% of the monomeric form. The active formessentially consists of the dimeric form, and contains only traces ofthe multimeric form. Stability tests underline the high stability duringstorage and during processing typical of the mutein in dimeric form.

DESCRIPTION OF THE FIGURES

FIG. 1: The figure shows the SDS-PAGE electrophoretic profile ofPLGF-1CG after re-suspension in physiologic solution at 20 mg/ml andstorage at 4-8° C. for 40 days. The profile is compared with the profilefor the mutein immediately after solubilisation.

(M) indicates the molecular weight references; (1) indicates thePlGF-1CG (3 micrograms) solubilised in the physiologic solution at aconcentration of 20 mg/ml and frozen (control); (2) indicates thePlGF-1CG (3 micrograms) solubilised in the physiologic solution at aconcentration of 20 mg/ml and stored at 4-8° C. for 40 days.

FIG. 2: The figure shows the data from table 2 expressed in graph form.

FIG. 3: The figure shows the SDS-PAGE electrophoretic profile of themutein PLGF-1CG solubilised in Carbopol gel at 0.2 mg/ml and stored at4-8° C. for 170 days. (M) indicates the molecular weight references; (1)indicates the PlGF-1CG (2 micrograms) solubilised in Carbopol gel at aconcentration of 0.2 mg/ml and stored at 4-8° C. for 170 days; (2)indicates the PlGF-1CG (2 micrograms) solubilised in Carbopol gel at aconcentration of 0.2 mg/ml and frozen (control).

FIG. 4: The figure shows the SDS-PAGE electrophoretic profile of thenative form of PLGF-1 solubilised in Carbopol gel at 0.2 mg/ml andstored at 4-8° C. for 150 days. (M) indicates the molecular weightreferences; (1) indicates the PLGF-1 (2 micrograms) solubilised inCarbopol gel at a concentration of 0.2 mg/ml and stored at 4-8° C. for150 days; (2) indicates the PlGF-1 (2 micrograms) solubilised inCarbopol gel at a concentration of 0.2 mg/ml and frozen (control).

FIG. 5: The figure is a schematic illustration of the process forconstruction of the plasmid pET3PLGF1CG, coding the PLGF-1 muteindenominated PLGF-1CG.

FIG. 6: The figure shows the angiogenic activity of wild type PLGF-1 andof the mutein PLGF-1CG at increasing concentrations of substance. Theactivity of the basic fibroblast growth factor bFGF is indicated as areference.

FIG. 7: The figure shows the effect of the mutein PLGF-1CG onisoprenaline-induced ischemic damage to cardiac tissue in rabbit. The xaxis indicates the days treatment and the value AUC representing thetotal area included within the curve identified by the daily ECG scores.

SEQUENCE LIST

SEQ ID NO:1 nucleotide sequence for wild type PLGF-1 without signalpeptide.

SEQ ID NO:2 nucleotide sequence for natural PLGF-1.

SEQ ID NO:3 sequence for the oligonucleotide used as forward primer inthe PCR.

SEQ ID NO:4 sequence for the oligonucleotide used as reverse primer inthe PCR.

DETAILED DESCRIPTION OF THE INVENTION

The complete polypeptide sequence of the human factor PLGF-1 of 149amino acids, along with a fragment of cDNA of 1645 nucleotidescomprising the sequence encoding the factor PLGF-1, are indicated in thepatent EP-B-0 550 519. A freely accessible plasmid containing thenucleotide sequence of 1645 bases has also been filed with ATCC underATCC filing number 40892.

The sequence encoding the pre-protein is comprised between positions 322and 768 and is indicated in this application as SEQ ID NO:1.

Wild type PLGF-1 in pre-protein form is a polypeptide with 149 aminoacids comprising a signal peptide of 18 amino acids in the N-terminalportion. The sequence for the mature protein, delimited by positions 19and 149, is indicated in this application as SEQ ID NO: 2. Said sequencecomprises 9 cysteine residues (Cys) located in positions 35, 60, 66, 69,70, 77, 111, 113 and 125.

In the muteins according to the invention, at least one of said cysteineresidues is eliminated or substituted by another residue, the onlycondition being that the mutation does not significantly affect oreliminate the ability of the mutein in its monomeric form to generatethe biologically active and therapeutically useful dimeric form.Experimental data has shown that the eliminated or substituted residuemust for preference be located in the C-terminal portion of the protein,and that the optimum residue for the purpose of the invention is the onein position 125.

Muteins of the wild type placental growth factor can be produced bysynthesis using polymer synthesis techniques known from the literature.However the preferred method is expression of the protein in geneticallymodified host cells. For this purpose the host cells are transformed byintroducing a cloning vector and/or an expression vector comprising aninsert that corresponds to the PLGF-1 gene after suitable modification.

Preparation of the DNA encoding the muteins according to the inventionis carried out by site-specific mutagenesis and implies point mutationin codons corresponding to cysteine, that is to say TGC or TGT. Thesemutations may be deletions or substitutions of one or more bases,without causing reading frame shift downstream of the mutation. In thecase of deletion a complete codon must therefore be removed. Preferably,site specific mutation is a point substitution of a base in a cysteinecodon, with consequent formation of a new codon. In this sense themutation will result in substitution of a cysteine residue with anotheramino acid residue.

Various known techniques of site-specific mutagenesis can be used toprepare the cDNA encoding the required mutein.

Methods that can be used are, for example, mutagenesis obtained witholigonucleotides (Adelman et al. “DNA” 2:183, 1983), PCR mutagenesis(Leung et al. Technique 1:11-15, 1989) or cassette-mutagenesis (Wells etal. Gene 34:315, 1985).

In the preferred embodiment of the invention, synthesis of the mutantDNA is carried out using the mutation technique through polymerase chainreaction (PCR). The cDNA encoding the wild type type PLGF-1 factordescribed in literature or any equivalents thereof caused bydegeneration of the genetic code was used as a template for the PCR.Preferably, only the portion encoding the methionilated protein in theN-terminal position with or without signal peptide will be used; forexample the sequence reported in the present application as SEQ ID NO:1or equivalents thereof, comprised in the expression vector pET3PlGF-1corresponding to the protein without the signal peptide. Theoligonucleotide 5′-3′ (forward) complementary to the region encoding theN-terminal portion of the protein, and the oligonucleotide 5′-3′(reverse) complementary to the region of the sequence comprising thecysteine codon to be mutated were used as primers for the PCR. Thelatter oligonucleotide will contain the base substitution orsubstitutions necessary to introduce the mutation required. The primersused may equally contain additional bases in the 5′ and/or 3′ terminalregions to introduce restriction sites suitable to isolate and purifythe mutated sequence.

The codon corresponding to the cysteine residue may be substituted by acodon encoding any neutral amino acid, whether polar such as Ser, Thr,Gln, or Asn, or non-polar such as Gly, Ala, Val, Ile or Leu. Preferredamino acids are Gly and Ala.

In the preferred embodiment the forward primer is represented by thesequence identified as SEQ ID NO: 3, while the reverse primer isrepresented by the sequence SEQ ID NO: 4. The latter comprises a T→Gsubstitution in position 382 of sequence SEQ ID NO: 1, a substitutionthat transforms the TGC codon of the cysteine in position 125 of thesequence SEQ ID NO:4 into a GGC codon corresponding to a glycine.

The suitably modified cDNA is subsequently cut and inserted into anexpression vector under the control of a suitable inducible systemcompatible with the host cell.

Preferably, inducible expression systems compatible with prokaryoticcells are used. Examples of these systems are:

-   -   pBAD expression system (In vitrogen BV) in which protein        synthesis is placed under the control of the araBAD promoter and        can be induced in various strains of E.coli using arabinose;    -   T7 Expression System (In vitrogen BV or Promega) in which        protein synthesis is controlled by the RNA polymerase promoter        for phage T7 and can be induced using lactose,        isopropyl-β-D-thiogalactopyranoside (IPTG) or derivatives or        functionally equivalent analogous thereof. In this case it is        necessary to use type DE3 (B121(DE3) or JM109(DE3)) derivatives        of E.coli, that is to say ones that contain a copy of the gene        for phage T7 Rna polymerase placed under the control of a        lactose-inducible promoter;    -   Trc expression system (In vitrogen BV)in which protein synthesis        is placed under the control of the hybrid promoter trc. This        promoter has been obtained by fusion of the trp promoter with        lac promoters and it can be induced in various strains of E.coli        by means of lactose or similar equivalents thereof(IPTG);    -   Tac expression system (Amersham biosciences) in which protein        synthesis is placed under the control of the tac promoter. In        this system, protein synthesis is induced in strains of E.coli        lacIq (type LM105) by means of lactose or similar equivalents        thereof(IPTG);    -   P_(L) expression system in which protein synthesis is placed        under the control of the P_(L) promoter and may be induced by        the addition of tryptophan. In this case the use of E.coli        derivatives (GI724) containing a copy of the gene encoding the        cI repressor of the Lambda phage is required, under the control        of a tryptophan-inducible promoter.

It is obviously possible to express the modified cDNA encoding themutein in eukaryotic host cells derived from yeast or from multicellularorganisms. In this case an expression system compatible with said cellswill be selected.

In the preferred embodiment of the invention expression is carried outunder the control of the T7 phage RNA polymerase system and induced withisopropyl-β-D-thiogalactopyranoside.

The expression vector also comprises additional sequences encoding thenormal functions necessary for cloning, selection and expression, suchas selective markers and/or a polyadenylation site and/or atranscription regulating sequence.

Host cells are therefore transformed, using standard techniques wellknown to the man skilled in the art, with the expression vectorcontaining the cDNA encoding the mutein required. These cells may beprokaryotic, eukaryotic, animal, human or plant cells, in particularbacterial cells, such as E.coli or Bacillus, yeast cells, such asSaccharomyces, or animal cells, such as Vero, HeLa, CHO, COS.

The preferred micro-organism is obtained by integration into thecommercially available strain [B12(DE3)pLysS] (Promega Corporation USA)of the gene from a human PLGF-1 mutein.

The modified cells used to produce the muteins according to theinvention are stored before use in lyophilised form to preserve theirexpression capacity. At the time of use, the lyophilised material isre-solubilised using a suitable buffer.

The modified host cells are then cultivated in liquid culture medium.Although a wide range of known culture mediums is commercially availableand can effectively be used, the fermentation step in accordance withthe invention is preferably carried out in a culture medium free fromany material of animal or human origin, in order to avoid any risk ofinfection. Yeast extracts (Difco) additioned with one or more suitableantibiotics represent the most suitable medium for the process. Thefermentation step may be preceded by a pre-inoculation step in which thelyophilised micro-organism is suspended in the culture medium andsubjected to consecutive incubation and dilution steps, aimed atobtaining an optimum quantity of micro-organism cells in the culture.

Fermentation is carried out in the culture medium seen above, at asuitable temperature for the micro-organism, normally approximately 37°C., in the presence of a percentage of dissolved O₂, with respect to thesaturation with air, of between 20% and 40%, preferably 30%. The pHduring culture is maintained at optimum values for the micro-organismbeing used, which will normally be neutral or very slightly acid oralkaline (6.4 to 7.4). Furthermore, given that the fermentation processtakes place under stirring, the use of anti-surfactants is advantageous.

As fermentation progresses it is accompanied by an increase in theoptical density of the culture medium. Therefore the optical density at600 nm is the parameter used according to the invention to monitor theprogress of the process. The cellular and therefore optical densityreached in the culture at the time that expression is induced must besufficiently high to guarantee a high yield of expressed protein.Although optical densities at 600 nm (OD⁶⁰⁰) higher than 0.2 can alreadybe used, optical densities of up to 50 can be obtained thanks to theculture mediums employed. Densities exceeding 18 are preferable in orderto obtain high mutein production levels. Densities of between 16 and 20have given optimum results. Fermentation is then maintained at theconditions seen above until these optical density values are reached,then expression of the protein is induced.

Any agent or chemical-physical condition capable of inducing theheterologous mutein expression mechanisms in the cells of themicro-organisms being used may be employed. In the specific case thatthe bacterial strain BL21(DE3)pLysS modified with an expression plasmidcontaining the T7 phage promoter is used, expression is induced withlactose or derivatives thereof, such asisopropyl-β-thiogalactopyranoside (IPTG) in a suitable concentration,that is to say approximately 1 mM. The duration of the induction mayvary according to requirements. Good results have been obtained forperiods of several hours, preferably from 3 to 4 hours; in the optimumprocess induction is maintained for 3 hours and 20 minutes using apercentage of dissolved O₂ equivalent to approximately 10%.

Samples of cells are taken prior to and following induction andsubjected to control analytical techniques such as SDS-PAGEelectrophoresis, to determine the results of the induction.

When expression of the protein has reached the levels required, thecells are separated from the culture medium, for example bycentrifugation, and subjected to an extraction process.

The extraction process foresees an initial cell lysis step. In effectwhen the mutein is expressed in bacteria, it remains segregated insidethe host cell, in the form of inclusion bodies. The lysis process may becarried out using freeze/thaw, French Press, ultrasound (sonication) orsimilar known techniques, in lysis solutions containing suitableconcentrations of surfactants, preferably containing Triton X100 inconcentrations of from 0.5% to 1%. The preferred technique when usingthe bacterial strain BL21(DE3)pLysS is the freeze/thaw technique, whichin an optimum embodiment is repeated for at least two consecutivecycles.

Given that the PLGF-1 mutein is released into the lysis medium asexpressed by the host cell, that is to say in the biologically inactivemonomeric form, lysis is followed by a renaturation step, at least inpart, consisting in dimerisation of the monomer. Renaturation of themutein is obtained by adding suitable concentrations ofoxidating-reducing pairs to the diluted solution, followed by anincubation period of from 10 to 30 hours, preferably 18 to 20 hours at atemperature of between 10° C. and 30° C., preferably 20° C. understirring. Examples of these pairs are: Cystine/Cysteine,Cystamine/Cysteamine, 2-hydroxyethyldisulfide/2-mercaptoethanol orglutathione in oxidised and reduced form. The latter represents thepreferred agent and is used, in oxidised form, at a concentration offrom 0.1 to 2.5 mM, preferably 0.5 mM and, in reduced form, from 0.25 to6.25 mM, preferably 1.25 mM.

In the case where the expressed PLGF-1 mutein is released into the lysismedium in the form of inclusion bodies, the renaturation step is recededby a solubilisation passage. The fraction containing the inclusionbodies is solubilised in a denaturing buffer containing know denaturingagents such as urea, guanidine isothiocyanate, guanidine hydrochloride.Preferably, the denaturing solution is a solution of urea in denaturingconcentration, for example 8M. To accelerate the solubilisation processit may be advantageous to subject the fraction containing the inclusionbodies to homogenisation or ultrasound (sonication). The solutioncontaining the protein is subsequently diluted with the denaturingbuffer itself and/or with a diluent solution until reaching an opticaldensity measured at 280 nm of approximately 0.5 OD²⁸⁰. Suitable dilutionsolutions contain salts and polyethylenglycol (PEG) and have an alkalinepH (pH approximately 8).

The release of the PLGF-1 mutein into the lysis medium is normallyaccompanied by the release of the various components and endocellularsubstances from the micro-organism, above all the nucleic material,which may affect or interfere with the protein purification process. Toavoid this problem, the suspension/solution obtained directly from celllysis may be subjected to an additional optional and preliminaryprocessing step consisting in fragmentation of said material. Thisresult is obtained by means of enzymatic agents, such as DNAses (naturalor recombinant such as Benzonase), chemical agents, such as deoxycholicacid, or physical-mechanical agents, such as ultrasound (sonication),rapid stirring with blades, for example in a blender. Physicalfragmentation of the DNA is carried out in suitable volumes of washingsolution containing chelating agents and detergents, for example EDTAand Triton X100, and is preferably repeated for a number of cycles,alternated with dilution, centrifugation and elimination of thesupernatant in order to remove every cellular component or substancefrom the fraction containing the inclusion bodies.

Although the PLGF-1 mutein after renaturation can already be used as is,it is preferably subjected to at least one purification step using anyone of the techniques well known to the man skilled in the art forpurification of proteic material. For this purpose, the mutein may besubjected to gel-filtration, ion exchange chromatography, affinitychromatography, HPLC, reverse phase chromatography and/or gelelectrophoresis. Preferred techniques are anionic exchange and reversephase chromatography. When it is necessary to obtain an extremely highlevel of purification, for example one suitable for therapeutic use, atleast two of the techniques mentioned above are combined in sequence.

The solution containing the partially renatured mutein, that is to sayat least in part in dimeric form, may be loaded onto anionic exchangeresin in order to enrich the mixture with the dimeric form and free itfrom bacterial contaminants. Any commercially available matrix suitablefor anionic exchange chromatography may be used, to the extent that itscapacity, load and flow speed characteristics are compatible with anindustrial process. In a preferred embodiment a high flow resin, forexample Q-sepharose Fast Flow (Amersham biosciences) or an equivalent,is used. The resins used allow ample volumes of proteic solution to beloaded, with a Load Volume/Column Volume ratios varying from 1:1 to10:1. Vol./Vol. ratios close to 10:1 are preferred, as they enable useof the column to be optimised. The entire chromatography process canadvantageously be carried out automatically by a computerised systemcontrolled by a suitable program, for example the FPCL Director Softwaresystem (Amersham biosciences).

In a variant of the process seen above, the partially renatured muteinmay be purified by reverse phase chromatography.

In this case, the suitably diluted solution containing the partiallyrenatured mutein, that is to say partly in dimeric form and partly inmonomeric form, can be loaded onto any commercially availablechromatography matrix suitable for the use indicated. Preferably, aresin is used that has a granulometry such as to guarantee optimumexploitation of the matrix absorption capacity together with easypacking of the column itself. Examples of these matrixes are the resinsRP Source 15 or RP Source 30 (Amersham biosciences). All the balancing,loading, resin washing and elution solutions are hydro-organic solutionscomprising different percentages of organic solvent. Examples of thesesolutions are solutions comprising ethanol, methanol or acetonitryl.Preferably, hydroalcoholic solutions comprising increasing percentagesof ethanol are used. Advantageously, the entire reverse phasechromatography process is carried out automatically by a computerisedsystem operating under the control of a suitable program, e.g. the FPCLDirector Software system (Amersham biosciences).

When two or more purification techniques are used one after the other,the mutein may be obtained in a highly purified active form, that is tosay with the protein comprised essentially in dimeric form, and withoutany contamination by the monomeric form. The product obtained in thisway comprises not less than 98.5% of the active form, preferably notless than 99.5%. The residual monomeric form does not exceed 1.5%. Giventhat the mutein is chemically stable, all multimerisation products arelimited to traces. The pure protein obtained according to the methoddescribed above may be subjected to further processing steps, forexample membrane ultrafiltration. In this case the product is filteredon a membrane with a filtration limit (cut-off) lower than or equal to30 kD and subjected to diafiltration against water acidulated with TFAuntil reaching a dilution factor of 1:10⁶. The final product obtained inthis way may be adequately formulated with lyophilisation additives andlyophilisate to preserve biological activity at optimum levels. In apractical embodiment of the invention, the mutein can be suitablyextracted, isolated and purified in accordance with the purificationprocess described in international patent application PCT/IT02/00065(Geymonat) to purify the wild type PLGF-1 protein, adapting theoperating conditions if necessary.

The chemical stability of PLGF-1 muteins according to the invention hasbeen evaluated in tests in which the lyophilised mutein and the wildtype PlGF-1 protein were solubilised in saline solution or formulated ina gel and stored at a temperature of 4-8° C. for periods of 40, 170 and210 days. The results reported below clearly indicate that the mutein inits active dimeric form is stable at all the concentrations analysed(20, 5 and 1 mg/ml), in that it does not tend to precipitate ormultimerise, indeed the concentration is found to remain at the initialvalues. On the contrary, the concentration of the dimeric form of thewild type protein decreases drastically after only a few days.

The muteins according to the invention show, along with an improvedchemical/biological stability, an angiogenic activity comparable withthat of the wild type PLGF-1 protein. This activity makes the PLGF-1muteins according to the invention suitable for all therapeutic andcosmetic applications of natural PLGF-1 currently known from the priorart.

The angiogenic action of the PLGF-1 muteins of the invention has beendetermined using known techniques, carried out in vivo or in vitro. Inparticular the rabbit cornea vascularization test or the chickenchorioallantoid membrane vascularization test (CAM) were used, asdescribed by Maglione et al. “Il Farmaco”, 55, 165-167 (2000).

A second method used to evaluate cutaneous vascularization iscomputerised morphometric analysis of samples of animal skin, asdescribed by Streit et al. (Proc. Natl. Acad. Sci. USA 1999, December21, 96(26), pages 14888-14893). Cutaneous sections isolated fromlaboratory animals, treated or untreated according to the presentinvention, were immuno-histochemically coloured using monoclonalanti-CD31 antibodies for the animal used. The sections treated in thisway were analysed under an electron microscope to evaluate the number ofblood vessels per mm2, their average dimensions and the relative areaoccupied by them. The angiogenic effect of the muteins on the myocardialtissue and in particular in the case of ischemia or myocardial infarctwas evaluated on an animal model of cardiac ischemia as described byMaglione et al. (supra).

The activity of the muteins according to the invention in treatment ofscleroderma was evaluated on an animal model as described by Yamamoto T.et al. in Arch. Dermatol. Res. November 2000, 292(11), pages 535 to 541.A state of scleroderma is induced in C3H mice with bleomycin (100mcg/ml) injected daily subcutaneously for 3 weeks. After 3 weeks, theanimals are sacrificed and samples of skin from the treated areas aresubjected to histological analysis. The effect of the treatmentunderlines histological events that can be attributed to cutaneoussclerotisation induced by the bleomycin and, in particular, skinthicknening and high hydroxiproline levels.

The results reported below underline the effectiveness of the PLGF-1muteins according to the invention in the treatment of all thosepathological or natural degenerative states that area subject toimprovement following an increase in vascularization of the areasinvolved. A first application is the preventive or curative treatment ofischemia and damage following ischemic events. Conditions liable to besuitable for treatment are ischemia of the myocardial tissue, myocardialinfarct, ischemic ictus and chronic myocardial diseases, cerebralischemia and ischemic ictus, intestinal ischemia, peripheral ischemia ofthe limbs. A second therapeutic application is treatment of scleroderma.This is a disease that involves the microvascular system, the cutaneousand subcutaneous connective tissue and the connective tissue of internalorgans. The disease induces activation of the fibroblasts and excessiveproduction and tissue and perivasal deposit of collagen, whichcontributes heavily towards the formation of fibrosis and calcificationareas, and therefore to the appearance of the symptoms induced by thedisease. In particular it can be seen under capillaroscopy that largeamounts of sclerotised collagen surround the skin vessels, causingrestriction of the vessel lumen. A circumscribed scleroderma withcutaneous involvement can be distinguished, characterised by hardeningand thickening of the skin due to excessive and inadequate deposit ofcollagen, and a progressive systemic scleroderma in which the bloodvessels are associated with the cutaneous fibrosis, along with asystemic sclerosis with lesions to the viscera. The skin, above all thatof the fingers and hands, is seen to be hardened, thickened andoedematous. The disease is also present at myocardial level with cardiacinsufficiency, and at pulmonary, gastrointestinal, renal andosteo-muscular system level. Some patients also develop erosivearthropathies induced by cutaneous fibrosis, which greatly complicatethe mobility of joints. Maglione et al. have reported (Italian patentapplication RM2002A000119) with regard to the wild type PLGF-1 factor,that the promotion of angiogenesis, in particular in the skin, as aresult of treatment with compositions containing PLGF-1 has a beneficialeffect on the general state of neomycin-induced sclerosis in mice. Thesame results were observed on animals in which a state of sclerodermahad previously been induced and treated with muteins according to theinvention.

A third therapeutic application is treatment supporting healingprocesses in burns, ulcers and internal or cutaneous wounds,particularly during the post-operative steps.

A further application according to the invention relates to treatment ofthe phenomena typical of skin ageing. This treatment, althoughconsidered essentially cosmetic, has therapeutic implications whentaking into consideration the precocious deterioration of cutaneoustissue due to prolonged exposure to sunlight (photo-ageing), toradiation of other types or to aggressive environmental/atmosphericagents.

Examination of samples of photo-damaged skin under an electronmicroscope reveals a typical microvascular morphology that ischaracterised, among other things, by the presence of capillaries thatare pathologically dilated and wrapped with elastin or surrounded by adense amorphous material. It has been observed that stimulation of newskin vascularization generates, in both naturally and precociously agedskin, a modulating effect on the extra-cellular matrix responsible forskin tone and thickness. The increased capillary vascularization isaccompanied by an increase in the fibroblasts and the production of newcollagen, followed by a general improvement in the appearance of theskin.

A further application of the compounds of the invention relates to hairloss.

In effect the improved skin vascularization, specifically in theperifollicular area, is accompanied by modulation of the growth ofcutaneous appendages (head hair, body hair, etc.) in the sense ofprevention of hair loss and promotion of its regeneration. The anagenicphase, which corresponds with the hair growth phase, is accompanied by anatural increase in vascularization of the hair follicle. The localangiogenic action promotes this vascular increase and the consequentgrowth of the hair. Computerised morphometric analysis of sections ofskin proximal to the piliferous follicles of animals treated with thecompositions of the invention have shown not only an increase in thedimensions of the hair lumen and hair density, and therefore a generalincrease in perifollicular vascularization, but also an increase in thedimensions of the hair bulb and the diameter of the hair itself.

The effect of preventing hair loss of promoting re-growth can be appliednot only in the case of natural loss, but also in the case of lossfollowing clinically significant states such as alopecia, hormonedisorders, chemotherapy, radiotherapy or medicaments administration.

The above identified therapeutic indications relate to direct, systemicor local administration of a mutein of the PLGF-1 factor. However, themuteins of the invention can also be used to produce antibodies,polyclonal, monoclonal or functionally active fragments capable ofrecognising the endogenous PLGF-1 factor, specifically those regions ofthe sequence of amino acids containing the bonding site for PLGF-1 andthe receptor. Antibodies of this type are capable of neutralising theangiogenic activity of PLGF-1 and find application in treatment of allthose conditions characterised by pathological angiogenesis. Examples ofthese conditions are inflammatory disorders such as rheumatoid arthritisor asthma, oedema, pulmonary hypertension and the formation anddevelopment of tumour tissue. The antibodies themselves can be used asimmuno-diagnostic reagents in methods for qualitative and quantitativedetermination of endogenous PLGF-1 production. These reagents findapplication in diagnosis of all those pathological states accompanied byabnormal production of PLGF-1, such as the formation and development oftumour tissue.

Antibodies or fragments thereof capable of recognising the PLGF-1muteins are prepared following techniques well known to the man skilledin the art, e.g. following the techniques described in applicationWO-A-01/85796 for the production of antibodies specific for the wildtype PLGF-1 protein.

The present invention likewise relates to pharmaceutical compositionscontaining the muteins described above or the corresponding neutralisingantibodies as active agents or as diagnostic reagents.

Any formulation suitable for systemic or local administration may beused according to the invention. In particular, the PLGF-1 factor may beadministered parenterally with a systemic or local effect, or topicallyon the skin or mucosa with a mainly local effect. A systemic effect ismainly obtained by intravenous administration, although intraperitonealor intramuscular administration is also possible. A local effect isobtained by topical, or intramuscular, subcutaneous, interarticularparenteral administration. The PLGF-1 muteins may likewise beadministered at local level by electrotransport of ionophoresis.Subcutaneous implants can likewise be used when delayed release isrequired over a period of time. Oral administration of the factor isalso possible, although it is less strongly recommended in view of thedelicate nature of the active product.

Compositions for systemic or local parenteral use include solutions,suspensions, liposome suspensions, W/O or O/W emulsions. Compositionsfor topical use include solutions, lotions, suspensions, liposomesuspensions, W/O, O/W, W/O/W, O/W/O emulsions, gels, ointments, cremes,pomades and pastes. In a preferred embodiment the active substance isformulated in lyophilised form, mixed with suitable lyophilisationadditives and ready to be resolubilised using therapeutically acceptablediluents. Lyophilisation additives that can be used are: buffers,polysaccharides, saccharose, mannitol, inositol, polypeptides, aminoacids and any other additive compatible with the active substance. In apreferred embodiment of the invention, the active substance is dissolvedin a phosphate buffer (NaH2PO4/H2O—Na2HPO4/2H2O) in an amount such thatthe mutein/phosphate ratio after lyophilisation is comprised between 1:1and 1:2. Suitable diluents for parenteral use are: water, salinesolution, sugar solutions, hydro-alcohol solutions, oily diluents,polyoils, such as glycerol, ethylene or polypropylene glycol, or anyother diluent compatible with the administration method in terms ofsterility, pH, ionic strength and viscosity.

In the case of emulsions or suspensions the composition may containsuitable surfactant agents of a non-ionic, zwitterionic, anionic orcationic type commonly used in the formulation of medications.Hydrophilic O/W or W/O/W emulsions are preferable forparenteral/systemic use, whereas lipofillic W/O or O/W/O emulsions arepreferable for local or topical use.

Furthermore, the compositions of the invention may contain optionaladditives such as isotonic agents, such as sugars or polyalcohols,buffers, chelating agents, antioxidants, antibacterial agents.

The compositions for topical use include liquid or semisolid forms. Theformer comprise solutions or lotions. These can be aqueous,hydro-alcoholic, such as ethanol/water or alcoholic and are obtained bysolubilisation of the lyophilised substance.

Alternatively, solutions of the active substance can be formulated asgels by addition of known gelling agents such as: starch, glycerine,polyethylene or polypropylene glycol, poly(meth)acrylate, isopropylalcohol, hydroxystearate, CARBOPOL®.

Other types of compositions for topical use are, emulsions orsuspensions in the form of pomades, pastes and creams. W/O emulsions arepreferred, as they provide faster absorption. Examples of lipophillicexcipients are: liquid paraffin, anhydrous lanolin, white vaseline,cetylic alcohol, stearylic alcohol, vegetable oils, mineral oils. Agentsthat increase the skin permeability, so as to facilitate absorption mayadvantageously be used. Examples of these agents are physiologicallyacceptable additives such as polyvinyl alcohol, polyethylenglycol ordimethylsulfoxide (DMSO).

Other additives used in the topical compositions are isotonic agents,such as sugars or polyalcohols, buffers, chelating agents, antioxidants,antibacterial agents, thickening agents, dispersing agents.

Compositions for local or systemic use with delayed release over aperiod of time may equally be used, and these include polymers such aspolylactate, poly(meth)acrylate, polyvinyl-pyrrolidone, methylcellulose,carboxymethylcellulose and other substances known in the art.Slow-release compositions in the form of subcutaneous implants based,for example, on polylactate or other biodegradable polymers may also beused.

Although the active substance is already stable in itself and ispreferably packed in lyophilised form, the pharmaceutical compositionsmay advantageously comprise additionally stabilising substances for thePLGF-1 muteins in the active dimeric form. Examples of these substancesare: Cysteine, Cysteamine, or glutathione.

Dosage of the mutein depends on the administration route and on theformulation chosen. For parenteral administration the amounts vary from1 mcg/Kg/day to 500 mcg/Kg/day, preferably from 10 mcg/Kg/day to 200mcg/Kg/day. These administrations are obtained with pharmaceuticalcompositions comprising from 50 mcg to 30 mg per unit dose, preferablyfrom approximately 500 mcg to 10 mg per dose. For therapeutic topicalapplication amounts varying from 0.1 mg to 10 mg per gram of compositionhave proved effective. Local cosmetic compositions for treatment of skinageing or hair loss comprises preferably from 0.01 mg to 0.09 mg ofactive substance per gram of composition.

The duration of the treatment varies according to the pathology or theeffect required. In the case of treatment of scleroderma the applicationperiod varies from 1 day to 12 months, according to the severity of thepathology. In the case of treatment for natural or precocious skinageing, the application period varies from 1 to 400 days, preferably forat least 30 days. In the case of treatment to prevent hair loss or topromote re-growth of hair the application period likewise varies from 1to 400 days.

The invention is described in the following by means of examples whosepurposes is solely illustrative and is not limiting.

EXAMPLE 1

Synthesis of the cDNA Encoding the Mutein.

The PlGF-1 MUTEIN, which we have called PlGF-1CG, was generated bymutation of the thymidine (T) No 382 (sequence SEQ ID NO:1) intoguanidine (G). In this way the TGC codon, nucleotides 382-384 of thesequence indicated above, encoding a cysteine was transformed into GGCencoding a glycine.

From an amino acid point of view the cysteine mutated into glycine inthe mutein PlGF-1CG is in position 125 of the sequence SEQ ID NO:2.

For synthesis of the DNA encoding the mutein, the PCR (polymerase chainreaction) technique was applied. The expression vector pET3PLGF1encoding the protein methionyl PlGF-1 without signal peptide (EP-A-0 550519) was used as a template for the PCR. In practice this is the wildtype PLGF-1 protein without the first 18 amino acids (signal peptide)and with a methionine in position 1 (N-terminal) (SEQ ID NO:2). Theoligonucleotides used as primers are the following:

-   -   oligo1 (forward primer) having the sequence 5′-CTGGC        GCATATGCTGCCTGCTGTGCCC-3═. This contains an NdeI site        (underlined) and the start codon (in bold);    -   oligo2 (reverse primer) having the sequence        5′-GGTTACCTCCGGGGAACAGCATCGCCGCCCC-3′. This contains a mutation        T→G (nucleotide underlined and in bold) that transforms the TGC        codon, encoding a cysteine, into the GGC codon (bold) encoding a        glycine.

The nucleotide chain obtained after PCR performed with a BioRad GeneCycler was subjected to a completion reaction (fill-in) and,subsequently, digested with the restriction enzyme NdeI. The fragmentobtained, with NdeI/“blunt” ends, was cloned in correspondingNdeI/“blunt” ends of the prokaryotic expression vector pET3 according tostandard protocols. In this way the plasmid pET3PLGF1CG encoding thePLGF-1 mutein known as PLGF-1CG was created.

This plasmid was used according to known techniques to transform thestrain of E. coli [B12(DE3)pLysS] (Promega Corporation USA) and producethe host strain [B12(DE3)pLysS PLGF-1CG].

The construction technique used for this plasmid is outlined in FIG. 5.

EXAMPLE 2 Production, Extraction and Purification of the Mutein PLGF-1CG

The micro-organism [Bl21(DE3)pLysS PLGF-1CG] has been cultivated in afermenter using as a culture medium the solution SBM comprising:Solution A (per 1 litre) Bacto yeast extract(Difco)   34 g Ammoniumsulphate  2.5 g Glycerol   100 ml H₂0 q.s. to:   900 ml Solution B (10X) (per 100 ml) KH₂PO₄  1.7 g K₂HPO₄—3H₂O   20 g or K₂HPO₄ 15.26 g H₂0q.s. to:   100 ml

Solutions A and B are mixed in sterile form at the time of use.

Expression is induced by means of IPTG(isopropyl-β-D-thiogalactopyranoside) 1 mM.

Fermentation is preceded by a pre-inoculation step. A tube oflyophilised micro-organism is taken and suspended in 1 ml of SBM+100μg/ml Ampicillin+34 μg/ml chloramphenicol, the suspension is furtherdiluted and incubated at 37° C. for one night. After dilution in thesame SBM solution additioned with Ampicillin and chloramphenicol, thepre-inoculate is divided into 4 Erlenmeyer flasks.

The content of each Erlenmeyer flask is incubated at 37° C. for 24hours. The contents of the 4 Erlenmeyer flasks are mixed and the OD⁶⁰⁰are read, diluting 1/20 in water (50 μl+950 μl water).

An established pre-inoculation volume is then centrifuged for 10 min. at7,500×g at 4° C. in sterile tubes. The bacteria are then re-suspended in20 ml SBM+200 μg/ml Ampicillin+10 μg/ml chloramphenicol per litre offermentation, by stirring at 420 rpm at R.T. for 20 minutes.

Fermentation is carried out in SBM solution containing 200 ug/mlampicillin, 10 μg/ml chloramphenicol and a suitable amount ofanti-foaming agent, at a temperature of 37° C. and in the presence of(30%) dissolved O₂ and at a pH value of between 6.4 and 7.4.

Induction is commenced when the culture medium has reached an opticaldensity at 600 nm (OD⁶⁰⁰) of between 16-20 units.

The inducing agent used is IPTG 1 mM in the presence of 10% dissolved O₂(with respect to air saturation). The duration of the induction isapproximately 3 hours. Induction is controlled by performing SDS-PAGEelectrophoresis, loading 20 μl of pre- and post-induction solutionpreviously boiled.

The culture medium containing the induced bacteria is then centrifugedat 7,500×g for 10 min. or at 3000×g for 25 min. at 4° C. and thesupernatant is discarded.

The bacterial cells are subsequently subjected to lysis followed byextraction and purification of the inclusion bodies. Bacterial lysis isperformed by means of 2 freezing/thawing cycles at −80/37° C. in a lysissolution containing 1 mM Mg₂SO₄+20 mM Tris-HCl pH8+1% Triton X100.

The lysis mixture is incubated at room temperature for 30 min. understirring (250 RPM), and then poured into a blender of suitable capacity,diluting with an amount of washing solution, containing 0.5% tritonX100+10 mM EDTA pH 8, equivalent to 3 ml per 450 OD⁶⁰⁰ of bacteria.

If necessary, 0.4 μl of undiluted anti-foaming agent are added for eachmillilitre of sample.

The solution is blended at maximum speed for 1 minute, or until thesample is homogeneous. The contents of the blender are then transferredto a container of suitable capacity, adequately diluted with 6.5 ml ofwashing solution for every 450 OD⁶⁰⁰ of bacteria, the suspensionobtained in this way is centrifuged at 13,000×g for 45 min. at 25° C.and the supernatant discarded.

The entire washing process is repeated for a number of cycles untilobtaining a final pellet containing the inclusion bodies of theexpressed protein.

Following this, the pellet containing the inclusion bodies issolubilised in 7 ml of denaturing buffer BD (8M urea, 50 mM Tris pH 8,Ethylendiamine 20 mM), then the solution is diluted to bring the finalurea concentration to 5M. Renaturation of the protein is carried out onthe solution obtained in this manner, by addition of reduced glutathione(final concentration equivalent to 1.25 mM) and oxidised glutathione(final concentration equivalent to 0.5 mM), and incubation at 20° C. for18-20 hours under stirring.

At the end of the incubation period it is centrifuged for 10 min. at 20°C., 10,000×g, and filtered through 0.45 or 0.8 μm filters.

The mutein in renatured form, i.e. in dimeric form, is subjected topurification by anion exchange chromatography.

The mutein solution is loaded onto Q-sepharose Fast Flow resin(Amersham-biosciences) equilibrated with buffer A (20 mMEthanolamine-HCl pH 8.5) and eluted, after washing, with 20% buffer B(buffer A+1M NaCl), corresponding to an NaCl concentration of 200 mM.

The partially purified mutein is brought up to a higher degree of purityby reverse phase chromatography. For this purpose the ion exchangechromatography elution peak is diluted with a solution containing TFAand ethanol so that the sample is diluted 1.5 times and contains 15%ethanol and 0.3% TFA. The addition of these substances enhances bondingof the mutein to the reverse phase resin.

The solution is loaded onto RP Source resin (Amersham-Bioscience) withan average diameter of 15 or 30 micron balanced with a solutioncontaining 40% Ethanol and 0.1% TFA. The washing solution removes themonomeric form of the mutein, whereas the dimeric form is eluted in anincreasing ethanol gradient until reaching a percentage of 70% ethanol.

The chromatography purification process is monitored controlling theabsorption at 280 nm of the eluted fractions.

The dimer solution obtained in this way is stored at −20° C., andsubsequently ultradiafiltered and lyophilised according to knowntechniques.

EXAMPLE 3 I Stability Study on the PLGF-1CG Mutein Carrying theSubstitution Cys 125 Gly

The PlGF-1CG mutein was solubilised in saline solution at theoreticalconcentrations of 20, 5 and 1 mg/ml. Simultaneously the PlGF-1 protein(without mutation) was also solubilised in saline solution at aconcentration of 20 mg/ml. All the samples were stored in therefrigerator (4-8° C.) for up to 40 days.

The actual concentration was determined by calculating the average ofthe values obtained from 3 suitable independent dilutions. The methodused was spectrophotometry using a wavelength of 280 nm and knowing thatan absorbency of 1 OD (optical density), measured a cuvette with astandard 1 cm optical path, corresponds to a concentration of PlGF-1 andPlGF-1CG equivalent to 1 mg/ml. During the days following the start ofthe experiment (time 0 on table 1), before carrying out the suitabledilutions to determine concentration, an aliquot of each sample wascentrifuged at 13,000 rpm in an ALC 4212 microcentrifuge for 10 minutesand the supernatant was used for subsequent analysis. In this way,possible precipitants were eliminated and, consequently, the resultsreferred to the protein remaining in the solution only.

The results of this study are illustrated in table 1, and clearly showthat the mutein, at all the concentrations analysed (20, 5 and 1 mg/ml),and at least up to 40 days, is stable in that it does not tend toprecipitate, so much so that the concentration is found to remain withinthe initial values. Vice versa, after just 4 days storage only 7.8% ofthe protein without mutation, stored at a concentration of 20 mg/ml atthe same conditions as the mutein, remains in the solution. This valuedrops still further (5.5%) after 12 days. It is important to note thateven after 24 hours of storage at 4-8° C. abundant precipitation of thePlGF-1 protein without mutation is already seen. TABLE 1 Time stored atAverage concentration (mg/ml) 4-8° C. PlGF-1CG PlGF-1CG PlGF-1CG PlGF-1(days) 20 mg/ml S.D. 5 mg/ml S.D. 1 mg/ml S.D. 20 mg/ml S.D. 0 20.381.00 5.43 0.04 1.07 0.02 20.14  0.18 4 19.69 0.57 5.25 0.27 0.98 0.081.57 0.10 12 20.46 0.54 5.28 0.19 1.00 0.01 1.11 0.01 26 20.72 0.33 N.A.N.A. N.A. 40 20.32 0.75 N.A. N.A. N.A.S.D. = Standard DeviationN.A. = Not Analysed

The electrophoresis profile (monomer-dimer-polymer composition) for thePlGF-1CG mutein (FIG. 1) is also substantially stable at the conditionsindicated above. In effect, SDS-PAGE electrophoresis in non-reducingconditions of the PlGF-1CG protein, solubilised in saline solution at 20mg/ml and frozen (control, line 1 of FIG. 1) or stored at 4-8° C. for 40days (sample, line 2 of FIG. 1), substantially reveals only minimumalterations, represented by disappearance of the tiny monomeric portionand by the formation of an extremely low amount of trimer (<0.5% of thedimer).

EXAMPLE 4 II Stability Study on the PLGF-1CG Mutein Formulated in Gel

The PlGF-1CG mutein and the non-mutated PlGF-1 protein were solubilisedat a concentration of 0.2 mg/ml in a gel is composed as follows:CARBOPOL 940 =   1% P/V Sodium Acetate pH 4.4 =      15 mM EDTA nonpHated disodium salt = 0.04% P/V Methyl paraben = 0.05% P/V

Brought up to, a pH of between 5.5 and 6 using NaOH/acetic acid.

The 2 gels were stored at 4-8° C. At various times a portion, induplicate, of the 2 gels (approximately 0.2 ml) was taken and weighed onthe analytical balances.

The weighed samples were additioned with a volume, expressed inmicrolitres, of non reducing 2× Sample Buffer equivalent to the weight,expressed in milligrams, of the gel portion.

After vortex stirring for 10 minutes and centrifuging at 13,000 rpm (ALC4214 microcentrifuge) for a further 10 minutes, the supernatant waswithdrawn. This was centrifuged again and the new supernatant wasremoved. A portion of the latter, together with quantity standards, wereanalysed by non reducing SDS-PAGE electrophoresis. After colouring, theelectrophoresis gels were analysed using a densiometer to find theconcentration of the dimeric form of the samples analysed.

The results obtained at various times and expressed as percentage dimerwith respect to that present at time zero, are indicated in Table 2 andexpressed in graph form in FIG. 2. From this data it can be seen that,whereas the mount of PlGF-1CG dimer remains constant until the end ofthe 170 days analysed, that of PlGF-1 drops to 71% after 150 days, andto 55% after 210 days.

The electrophoresis profile (monomer-dimer-polymer composition) alsoshows that the PlGF-1CG mutein is substantially sable at the conditionsindicated above (FIG. 3). In effect, SDS-PAGE electrophoresis innon-reductive conditions of the PlGF-1CG protein, solubilised incarbopol at 0.20 mg/ml and frozen (control, line 2 of FIG. 3) or storedat 4-8° C. for 170 days (sample, line 1 of FIG. 3), does not reveal anypolymerisation phenomena. Vice versa, these multimerisation phenomenaare clearly evident for the non-mutated PlGF-1 protein, as illustratedin FIG. 4 (compare line 1—protein stored at 4°-8° C. for 150 days, withline 2—frozen protein). TABLE 2 % dimer Time stored at GEL PlGF-1CG 4-8°C. (days) (mutein) GEL PlGF-1  0 100.00 100.00   30 108.00 N.A.  57 N.A.83.40 119 N.A. 72.70 133  98.70 N.A. 150 N.A. 71.10 170 101.50 N.A. 210N.A. 54.90N.A. = not analysed

EXAMPLE 5 Evaluation of the angiogenic activity of the PLGF-1CG mutein

The antiogenic activity of the PLGF-1CG mutein, of the wild type PLGF-1factor and, as a positive reference, of the basic fibroblast growthfactor (bFGF) were compared using the chicken chorioallantoid membranevascularization test (CAM) already described by Maglione et al. (“IlFarmaco” supra). Various amounts of mutein and wild type factor (between0 and 3 mcg/sponge) were absorbed on 1 mm³ gelatine sponges,subsequently implanted on the surface of CAMs. After 12 days, the CAMregions in contact with the samples were sectioned, coloured and theangiogenic effect was quantified using the morphometric technique knownas “point counting”. Specifically, the CAM sections were analysed undera microscope on a grid with 144 intersection points and the results wereexpressed as the percentage of the intersection points occupied by thecapillaries on a transversal section (percentage of the area that isvasculised). The results, illustrated in FIG. 6, show essentiallyequivalent angiogenic activity for the mutein and the wild type factor.

EXAMPLE 6 Evaluation of the Effect of the PLGF-1CG Mutein onIsoprenaline-Induced Cardiac Ischemia

The effect of the PLGF-1CG mutein on cardiac ischemia and infarct wasevaluated on ischemia induced in an animal model by means ofisoprenaline, as described by Maglione et al. (supra) in relation to thewild type factor. The experiment was carried out on rabbits, which weretreated with a single daily dose of 160 mcg/Kg of mutein or withequivalent volumes of excipient only, administered intravenously on days1 to 5. The Isoprenaline was administered subcutaneously on days 1 and2. The characteristics typical of the electrocardiogram (ECG) indicatingthe main ischemic damage, such as inversion of the T wave, widening ofthe S wave and prominence of the Q wave, are decidedly more marked inanimals treated with the excipient alone, with respect to animalstreated with the mutein under examination. Variations in the ECG oftreated and untreated animals were evaluated on a point scale rangingfrom zero to six, as reported below: 0, no lesion; 1, prominence of theS wave; 2, prominence of the T wave; 3, depression of the descending armof the T wave; 4, widening of the S wave; 5, inversion of the T wave; 6,prominence of the Q wave. The total area under the curve defined by theECG points during the 5 days of the test was likewise calculated fortreated and untreated animals. The results are illustrated in FIG. 7 andshow a significant reduction in the ischemic damage in animals treatedwith the PLGF-1CG mutein. The results underlined by theelectrocardiographic profile were confirmed by macro and microscopicobservation of the ischemic tissues. Said examination shows the presenceof ischemic lesions and histological alterations of moderated severitywith respect to the ones observed in the cardiac tissue of animalstreated with the excipient only.

EXAMPLE 7 Evaluation of the Effect of the PLGF-1CG Mutein onNeomycin-Induced Scleroderma

In this study the animal scleroderma model described by Yamamoto et al.(supra) was used.

A first group of C3H mice was treated with bleomycin (100 mcg/ml)injected daily subcutaneously for 3 weeks. Three other groups of C3Hmice were likewise treated as above, but 0.1, 1 and 10 mcg/ml of thePlGF-1CG mutein was added to the daily injection, respectively. After 3weeks treatment, the animals were sacrificed and samples of skin fromthe treated areas were taken and subjected to histological analysis. Theeffect of the treatment with PlGF-1CG at 1 and 10 mcg/ml, but not at 0.1mcg/ml, underlines a significant reduction in histological events thatcan be attributed to cutaneous sclerotisation induced by the bleomycin.In particular, skin thicknening and hydroxiproline levels weresignificantly decreased with respect to the mice treated with bleomycinalone.

EXAMPLE 8 Pharmaceutical Compositions

i) Solution for Parenteral Use:

58 milligrams of lyophilised mutein, containing 25 mg pure FLGF-1CG and33 mg phosphate buffer (10 mg NaH2PO4/H2O and 23 mg Na2HPO4/2H2O), andapproximately 125 ml saline solution for parenteral use, are packedseparately in vials prepared to allow mixing of the lyophilised productwith the diluent immediately prior to use. The concentration of activesubstance resulting after solubilisation is approximately 0.2 mg/ml.

ii) Topical Composition in Gel Form:

An amount of lyophilised substance containing 10 mg active substance iscarried in 20 ml of 10% ethanol hydro-alcoholic solution containing 20%DMSO. The solution is then additioned with a suitable gel excipientcontaining the following ingredients: 1% Carbopol 940, sodium acetate 15mM (pH 4.4), 0.04% p/v disodium EDTA, 0.05% p/v methyl paraben with afinal pH comprised between 5.5 and 6.

1-51. (canceled)
 52. A mutein of the monomeric form of human or animal type 1 Placental Growth Factor (PLGF-1) comprising the substitution or the elimination of at least one cysteine residue (Cys) in the C-terminal portion of the wild type protein polypeptide sequence said substitution or elimination does not affect the formation of the biologically active dimer, but prevents the multimerisation of said monomeric form.
 53. The mutein according to claim 52 characterised in that it involves the substitution or the elimination of the Cys residue in position 142 of the pre-protein polypeptide sequence, corresponding to cysteine in position 125 of SEQ ID NO:
 2. 54. The mutein according to claim 53 characterised in that the Cys residue in position 142 is replaced by a glycine residue (Gly).
 55. The mutein according to claim 52 in the form of a pre-protein or mature protein.
 56. The Mutein according to claim 53 comprising the elimination or substitution or addition of one or more amino acids of the wild type protein without altering the functional characteristics of the mutein.
 57. The mutein according to claim 53 that is at least 98.5% in dimeric form.
 58. A nucleotide sequence comprising the DNA encoding the PLGF-1 mutein according to claim
 53. 59. The nucleotide sequence according to claim 58 wherein a TGC or a TGT codon in the sequence encoding the wild type PLGF-1 is eliminated or modified.
 60. The nucleotide sequence according to claim 59 in which the thymidine base in position 382 of the sequence SEQ ID NO:1, or derivatives thereof caused by degeneration of the DNA, is substituted by a guanidine base.
 61. An expression system comprising the nucleotide sequence according to claim 58, flanked by untranslated sequences for control and regulation of expression.
 62. The expression system according to claim 61, that is an expression system inducible in bacterial cells.
 63. The expression system according to claim 62, wherein the expression is under the control of an inducible promoter.
 64. The expression system according to claim 61 that comprises a T7 phage RNA-polymerase expression system, and wherein expression is induced by means of lactose, isopropyl-β-D-thiogalactopyranoside (IPTG) or a functional equivalent.
 65. A host cell transformed by means of the expression system according to claim
 61. 66. The cell according to claim 65, that is a bacterial cell.
 67. The bacterial cell according to claim 66 that is derived from a strain of E. coli.
 68. A process for the production of the nucleotide sequence according to claim 58 comprising producing the DNA encoding the PLGF-1 mutein by polymerase chain reaction (PCR), wherein the oligonucleotide of sequence SEQ ID NO:3 is used as a 5′-3′ (forward) primer and the oligonucleotide of sequence SEQ ID NO:4 is used as a 5′-3′ (reverse) primer.
 69. The process according to claim 68 in which the DNA sequence encoding the PLGF-1 protein without the signal protein is used as a template in the polymerase chain reaction.
 70. A process for the production and extraction of a mutein of the factor PLGF-1 comprising: cultivating the host cells according to claim 65 in a suitable culture medium, inducing the expression of the protein using a suitable inducer, isolating and lysing the cells and extracting the mutein from the lysis mixture.
 71. The process according to claim 70 comprising culturing the cells, until reaching an optical density (O.D.) of the culture medium between 0.2 and 50 units, at 600 nm before inducing the expression.
 72. The process according to claim 71 further comprising isolating the inclusion bodies released during lysis, solubilizing the inclusion bodies in a denaturing buffer and optionally homogenizing or sonicating.
 73. The process according to claim 72, further comprising diluting the solution of soluilized inclusion bodies and renaturing the proteic material in dimeric form by addition to the solution an oxydating-reducing agent.
 74. The process according to claim 73, further comprising at least one purification step selected from the group comprising ion exchange chromatography, reverse phase chromatography and ultrafiltration.
 75. A mutein according to claim 57 for use in a therapeutic treatment method.
 76. The mutein according to claim 75 in the treatment of ischemic diseases.
 77. The mutein according to claim 76 in the treatment of ischemia of the myocardial tissue, myocardial infarct, ischemic ictus and chronic ischemic myocardial diseases cerebral ischemia and ischemic ictus, intestinal ischemia, periferal ischemia of the limbs.
 78. The mutein according to claim 75 in the treatment of scleroderma.
 79. The mutein according to claim 75 in the treatment of skin ulcers, wounds, bums, post-operative treatment.
 80. The mutein according to claim 75 in the treatment of natural or precocious ageing of the cutaneous tissues.
 81. The mutein according to claim 75 in the treatment of natural or pathological hair loss.
 82. A Polyclonal antibody, a monoclonal antibody, or a functionally active fragment thereof, capable of recognising the mutein according to claim 57, capable of cross-reacting with the wild type PLGF-1 and of neutralising its angiogenic activity.
 83. The antibody according to claim 82 as a medicament to neutralise the angiogenic activity of endogenous PLGF-1 in the treatment of pathological forms accompanied by aberrant production of PLGF-1.
 84. The antibody according to claim 82 in the treatment of forms of tumour.
 85. The antibody according to claim 82 as reagent in a method for the diagnosis of pathological forms accompanied by aberrant production of endogenous PLGF-1.
 86. A pharmaceutical composition comprising the mutein according to claim 57 and a pharmaceutically acceptable excipient.
 87. A pharmaceutical composition comprising the antibody according to claim 82 and a pharmaceutically acceptable excipient.
 88. A Pharmaceutical composition according to claim 86 for parenteral, topical, oral, nasal or implant use.
 89. A Pharmaceutical composition according to claim 87 for parenteral, topical, oral, nasal or implant use.
 90. A Cosmetic composition containing the mutein according to claim 57 and a cosmetically acceptable excipient.
 91. A method for the preparation of the pharmaceutical composition according to claim 86, comprising associating the PLGF-1 mutein with a pharmacologically acceptable excipient and with other usual additives.
 92. A method for the preparation of the cosmetic composition according to claim 90, comprising associating the PLGF-1 mutein with a cosmetically acceptable excipient and with other usual additives. 